Polynucleotide encoding acyl-CoA synthetase homolog and use thereof

ABSTRACT

The present invention relates to an acyl-CoA synthetase homolog protein from microorganisms of the genus  Mortierella , a polynucleotide encoding the protein, and so on. The invention provides polynucleotides comprising an acyl-CoA synthetase homolog protein gene from, e.g.,  Mortierella alpina , expression vectors comprising these polynucleotides and transformants thereof, a method for producing lipids or fatty acids using the transformants, food products containing the lipids or fatty acids produced by the method, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/574,026, filed Jul. 19, 2012, now U.S. Pat. No. 9,289,007, which isthe National Stage of International Application No. PCT/JP2011/052035,filed Feb. 1, 2011, which claims priority to Japanese Patent ApplicationNo. 2010-019967, filed Feb. 1, 2010. The disclosure of application Ser.Nos. 13/574,026 and PCT/JP2011/052035 are expressly incorporated byreference herein in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 24, 2015, isnamed P42026_SL.txt and is 294,294 bytes in size.

TECHNICAL FIELD

The present invention relates to a polynucleotide encoding an acyl-CoAsynthetase homolog and use thereof.

BACKGROUND ART

Fatty acids containing two or more unsaturated bonds are collectivelyreferred to as polyunsaturated fatty acids (PUFAs) and known tospecifically include arachidonic acid (ARA), dihomo-γ-linolenic acid(DGLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc.Some of these polyunsaturated fatty acids cannot be synthesized in theanimal body. It is therefore necessary to compensate thesepolyunsaturated fatty acids as essential amino acids from food.

Polyunsaturated fatty acids are widely distributed; for instance,arachidonic acid can be separated from lipids extracted from the adrenalglands and livers of animals. However, polyunsaturated fatty acidscontained in animal organs are only in a small quantity and cannot beobtained sufficiently for large supplies when simply extracted orseparated from animal organs. For this reason, microbial techniques havebeen developed for obtaining polyunsaturated fatty acids by cultivationof various microorganisms. Above all, microorganisms of the genusMortierella are known to produce lipids containing polyunsaturated fattyacids such as arachidonic acid and the like.

Other attempts have also been made to produce polyunsaturated fattyacids in plants. Polyunsaturated fatty acids constitute storage lipidssuch as triacylglycerols and are known to be accumulated withinmicroorganism mycelia or plant seeds.

Acyl-CoA synthetase (ACS) is an enzyme catalyzing the thioesterificationof fatty acids and coenzyme A (CoA) and catalyzes the followingreaction.Fatty acid+CoASH+ATP→Acyl-CoA+AMP+PPi

Acyl-CoA produced by ACS is involved in various life phenomena includingthe biosynthesis and remodeling of lipids, energy production byβ-oxidation, acylation of proteins, expression regulation by fattyacids, etc. Furthermore, ACS is reportedly associated with extracellularuptake of fatty acids, intracellular transport of fatty acids, etc.(Non-Patent Documents 1 and 2). In view of the foregoing, it isconsidered to control the activity of ACS when polyunsaturated fattyacids or the like are produced by utilizing microorganisms or plants.

In the yeast Saccharomyces cerevisiae used as a model eukaryote, six (6)acyl-CoA synthetase genes (ScFAA1, ScFAA2, ScFAA3, ScFAA4, ScFAT1 andScFAT2) are known (Non-Patent Document 1). The proteins encoded by thesegenes are different in substrate specificity, timing of expression,intracellular localization and function.

Patent Document 1 discloses nine (9) genes as the acyl-CoA synthetasegene (ScACS) derived from Schizochytrium sp. Patent Document 1 alsodiscloses an increased production of DPA (n-6) (docosapentanoic acid(n-6)) or DHA when the gene encoding the Schizochytrium sp. PUFAsynthase system is co-expressed with ScACS, as compared to the casewhere the co-expression with ScACS is not involved.

In addition, acyl-CoA synthetase genes derived from animals and plantsare also reported (Non-Patent Document 2 and Patent Document 2).

-   [Patent Document 1] Japanese Unexamined Patent Application    Publication (Translation of PCT Application) No. 2009-529890-   [Patent Document 2] PCT International Publication Pamphlet WO    0209295-   [Non-Patent Document 1] B. B. A. 1771, 286-298, 2007-   [Non-Patent Document 2] Exp. Biol. Med., 233 (5), 507-521, 2008

DISCLOSURE OF THE INVENTION

Under the foregoing circumstances, it has been desired to isolate anovel gene that increases the amount of the fatty acids produced in ahost cell or changes the composition of fatty acids produced, when thegene is expressed in the host cell.

As a result of extensive investigations, the present inventors havesucceeded in cloning a gene encoding an ACS homolog of lipid-producingfungus Mortierella alpina (hereinafter “M. alpina”) (MaACS), andaccomplished the present invention. That is, the present inventionprovides the following polynucleotides, proteins, expression vectors,transformants, and a method for producing lipids or lipid compositionsand foods, etc. using the transformants, as well as foods produced bythe method, etc.

That is, the present invention is characterized as follows.

[1] A polynucleotide according to any one selected from the groupconsisting of (a) to (e) below:

(a) a polynucleotide comprising any one nucleotide sequence selectedfrom the group consisting of the nucleotide sequences shown by SEQ IDNOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51 and 56;

(b) a polynucleotide encoding a protein consisting of any one amino acidsequence selected from the group consisting of the amino acid sequencesshown by SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52 and 57;

(c) a polynucleotide encoding a protein consisting of an amino acidsequence wherein 1 to 100 amino acids are deleted, substituted, insertedand/or added in any one amino acid sequence selected from the groupconsisting of the amino acid sequences shown by SEQ ID NOs: 2, 7, 12,17, 22, 27, 32, 37, 42, 47, 52 and 57, and having an acyl-CoA synthetaseactivity or an activity of increasing the amount or changing thecomposition, of the fatty acids produced in a host cell when expressedin the host cell;

(d) a polynucleotide encoding a protein having an amino acid sequencehaving at least 60% identity to any one amino acid sequence selectedfrom the group consisting of the amino acid sequences shown by SEQ IDNOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52 and 57, and having anacyl-CoA synthetase activity or an activity of increasing the amount orchanging the composition, of the fatty acids produced in a host cellwhen expressed in the host cell; and,

(e) a polynucleotide which hybridizes to a polynucleotide consisting ofa nucleotide sequence complementary to any one nucleotide sequenceselected from the group consisting of the nucleotide sequences shown bySEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51 and 56 understringent conditions, and which encodes a protein having an acyl-CoAsynthetase activity or an activity of increasing the amount or changingthe composition, of the fatty acids produced in a host cell whenexpressed in the host cell.

[2] The polynucleotide according to claim 1, which is either one definedin (f) or (g) below:

(f) a polynucleotide encoding a protein consisting of an amino acidsequence wherein 1 to 10 amino acids are deleted, substituted, insertedand/or added in any one amino acid sequence selected from the groupconsisting of the amino acid sequences shown by SEQ ID NOs: 2, 7, 12,17, 22, 27, 32, 37, 42, 47, 52 and 57, and having an acyl-CoA synthetaseactivity or an activity of increasing the amount or changing thecomposition, of the fatty acids produced in a host cell when expressedin the host cell; and,

(g) a polynucleotide encoding a protein having an amino acid sequencehaving at least 90% identity to any one amino acid sequence selectedfrom the group consisting of the amino acid sequences shown by SEQ IDNOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52 and 57, and an acyl-CoAsynthetase activity or an activity of increasing the amount or changingthe composition, of the fatty acids produced in a host cell whenexpressed in the host cell.

[3] The polynucleotide according to [1] above, comprising any onenucleotide sequence selected from the group consisting of the nucleotidesequences shown by SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51and 56.

[4] The polynucleotide according to [1] above, encoding a proteinconsisting of any one amino acid sequence selected from the groupconsisting of the amino acid sequences shown by SEQ ID NOs: 2, 7, 12,17, 22, 27, 32, 37, 42, 47, 52 and 57.

[5] The polynucleotide according to any one of [1] to [4] above, whichis a DNA.

[6] A protein encoded by the polynucleotide according to any one of [1]to [5] above.

[7] A vector comprising the polynucleotide according to any one of [1]to [5] above.

[8] A non-human transformant, into which the polynucleotide according toany one of [1] to [5] above, or the vector according to [7] above isintroduced.

[9] A method for producing a lipid or fatty acid composition, whichcomprises collecting the lipid or fatty acid composition from theculture of the transformant according to [8] above.

[10] The method according to [9] above, wherein the lipid is atriacylglycerol.

[11] The method according to [9] above, wherein the fatty acid is apolyunsaturated fatty acid having at least 18 carbon atoms.

[12] A food product, pharmaceutical, cosmetic or soap comprising thelipid or fatty acid composition obtained by the production methodaccording to [9] above.

The polynucleotide of the present invention can be used fortransformation of an appropriate host cell. The transformant thusproduced can be used to produce fatty acid compositions, food products,cosmetics, pharmaceuticals, soaps, etc.

More specifically, the transformant of the present invention provides anextremely high production efficiency of lipids and fatty acids.Accordingly, the present invention can be effectively used tomanufacture pharmaceuticals or health foods which require a largequantity of lipids or fatty acids.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the correspondence between the cDNA sequence of MaACS-1(SEQ ID NO: 4) and putative amino acid sequence of MaACS-1 (SEQ ID NO:2).

FIG. 2A shows the alignment between the genome sequence of MaACS-1 (SEQID NO: 5) and CDS sequence of MaACS-1 (SEQ ID NO: 3).

FIG. 2B is a continuation from FIG. 2A.

FIG. 3A shows the correspondence between the cDNA sequence of MaACS-2(SEQ ID NO: 9) and putative amino acid sequence of MaACS-2 (SEQ ID NO:7).

FIG. 3B is a continuation from FIG. 3A.

FIG. 4A shows the alignment between the genome sequence of MaACS-2 (SEQID NO: 10) and CDS sequence of MaACS-2 (SEQ ID NO: 8).

FIG. 4B is a continuation from FIG. 4A.

FIG. 4C is a continuation from FIG. 4B.

FIG. 5 shows the correspondence between the cDNA sequence of MaACS-3(SEQ ID NO: 14) and putative amino acid sequence of MaACS-3 (SEQ ID NO:12).

FIG. 6A shows the alignment between the genome sequence of MaACS-3 (SEQID NO: 15) and CDS sequence of MaACS-3 (SEQ ID NO: 13).

FIG. 6B is a continuation from FIG. 6A.

FIG. 7A shows the correspondence between the cDNA sequence of MaACS-4(SEQ ID NO: 19) and putative amino acid sequence of MaACS-4 (SEQ ID NO:17).

FIG. 7B is a continuation from FIG. 7A.

FIG. 8A shows the alignment between the genome sequence of MaACS-4 (SEQID NO: 20) and CDS sequence of MaACS-4 (SEQ ID NO: 18).

FIG. 8B is a continuation from FIG. 8A

FIG. 8C is a continuation from FIG. 8B.

FIG. 9A shows the correspondence between the cDNA sequence of MaACS-5(SEQ ID NO: 24) and putative amino acid sequence of MaACS-5 (SEQ ID NO:22).

FIG. 9B is a continuation from FIG. 9A.

FIG. 10A shows the alignment between the genome sequence of MaACS-5 (SEQID NO: 25) and CDS sequence of MaACS-5 (SEQ ID NO: 23).

FIG. 10B is a continuation from FIG. 10A.

FIG. 11A shows the correspondence between the cDNA sequence of MaACS-6(SEQ ID NO: 29) and putative amino acid sequence of MaACS-6 (SEQ ID NO:27).

FIG. 11B is a continuation from FIG. 11A.

FIG. 12A shows the alignment between the genome sequence of MaACS-6 (SEQID NO: 30) and CDS sequence of MaACS-6 (SEQ ID NO: 28).

FIG. 12B is a continuation from FIG. 12A.

FIG. 13 shows the correspondence between the cDNA sequence of MaACS-7(SEQ ID NO: 34) and putative amino acid sequence of MaACS-7 (SEQ ID NO:32).

FIG. 14A shows the alignment between the genome sequence of MaACS-7 (SEQID NO: 35) and CDS sequence of MaACS-7 (SEQ ID NO: 33).

FIG. 14B is a continuation from FIG. 14A.

FIG. 15A shows the correspondence between the cDNA sequence of MaACS-8(SEQ ID NO: 39) and putative amino acid sequence of MaACS-8 (SEQ ID NO:37).

FIG. 15B is a continuation from FIG. 15A.

FIG. 16A shows the alignment between the genome sequence of MaACS-8 (SEQID NO: 40) and CDS sequence of MaACS-8 (SEQ ID NO: 38).

FIG. 16B is a continuation from FIG. 16A.

FIG. 16C is a continuation from FIG. 16B.

FIG. 17 shows the correspondence between the cDNA sequence of MaACS-9(SEQ ID NO: 44) and putative amino acid sequence of MaACS-9 (SEQ ID NO:42).

FIG. 18A shows the alignment between the genome sequence of MaACS-9 (SEQID NO: 45) and CDS sequence of MaACS-9 (SEQ ID NO: 43).

FIG. 18B is a continuation from FIG. 18A.

FIG. 19A shows the correspondence between the cDNA sequence of MaACS-10(SEQ ID NO: 49) and putative amino acid sequence of MaACS-10 (SEQ ID NO:47).

FIG. 19B is a continuation from FIG. 19A.

FIG. 20A shows the alignment between the genome sequence of MaACS-10(SEQ ID NO: 50) and CDS sequence of MaACS-10 (SEQ ID NO: 48).

FIG. 20B is a continuation from FIG. 20A.

FIG. 20C is a continuation from FIG. 20B.

FIG. 21A shows the correspondence between the cDNA sequence of MaACS-11(SEQ ID NO: 54) and putative amino acid sequence of MaACS-11 (SEQ ID NO:52).

FIG. 21B is a continuation from FIG. 21A

FIG. 22A shows the alignment between the genome sequence of MaACS-11(SEQ ID NO: 55) and CDS sequence of MaACS-11 (SEQ ID NO: 53).

FIG. 22B is a continuation from FIG. 22A.

FIG. 23A shows the correspondence between the cDNA sequence of MaACS-12(SEQ ID NO: 59) and putative amino acid sequence of MaACS-12 (SEQ ID NO:57).

FIG. 23B is a continuation from FIG. 23A

FIG. 24A shows the alignment between the genome sequence of MaACS-12(SEQ ID NO: 60) and CDS sequence of MaACS-12 (SEQ ID NO: 58).

FIG. 24B is a continuation from FIG. 24A

FIG. 25A shows the alignment between MaACS having relatively high aminoacid sequence homology to S. cerevisiae-derived FAA protein (FAA: fattyacid activation) and the FAA protein (SEQ ID NOS: 12, 17, 22, 27, 37,47, 52, 57, and 124-127, in order of appearance). The single underlinedand double underlined sequences denote the ATP-AMP motif and theFACS/VLACS-FATP motif, respectively.

FIG. 25B is a continuation from FIG. 25A.

FIG. 25C is a continuation from FIG. 25B.

FIG. 26A shows the alignment between MaACS having relatively high aminoacid sequence homology to S. cerevisiae-derived FAT protein (FAT: fattyacid transferase) and the FAT protein (SEQ ID NOS: 2, 7, 42, 32, and128-129, in order of appearance). The single underlined and doubleunderlined sequences denote the ATP-AMP motif and the FACS/VLACS-FATPmotif, respectively.

FIG. 26B is a continuation from FIG. 26A.

FIG. 27A and FIG. 27B show changes with the passage of time in lipidproduction (FIG. 27A) and arachidonic acid production (FIG. 27B), permycelia in MaACS-10-overexpressed M. alpina.

FIG. 28A and FIG. 28B show changes with the passage of time in lipidproduction (FIG. 28A) and arachidonic acid production (FIG. 28B), permycelia in MaACS-11-overexpressed M. alpina.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail. Theembodiments described below are intended to be presented by way ofexample merely to describe the invention but not limited only to thefollowing embodiments. The present invention may be implemented invarious ways without departing from the gist of the invention.

All of the publications, published patent applications, patents andother patent documents cited in this application are herein incorporatedby reference in their entirety. This application hereby incorporates byreference the contents of the specification and drawings in the JapanesePatent Application (No. 2010-19967) filed Feb. 1, 2010, from which thepriority was claimed.

As will be later described in detail in EXAMPLES below, the presentinventors have succeeded for the first time in cloning the full-lengthcDNA of lipid-producing fungus M. alpina-derived ACS homolog genes(MaACS-1˜12). The present inventors have also identified the nucleotidesequences of genomic DNAs of MaACS-1˜12 from M. alpina and putativeamino acid sequences thereof. The ORF sequences, putative amino acidsequences, CDS sequences, cDNA sequences and genome sequences ofMaACS-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 are SEQ ID NOs: 1, 6, 11,16, 21, 26, 31, 36, 41, 46, 51 and 56 (hereinafter these sequences arecollectively referred to as “ORF sequences of MaACS-1˜12”), SEQ ID NOs:2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52 and 57 (hereinafter thesesequences are collectively referred to as “amino acid sequences ofMaACS-1˜12”), SEQ ID NOs: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53 and58 (hereinafter these sequences are collectively referred to as “CDSsequences of MaACS-1˜12”), SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, 39, 44,49, 54 and 59 (hereinafter these sequences are collectively referred toas “cDNA sequences of MaACS-1˜12”) and SEQ ID NOs: 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55 and 60 (hereinafter these sequences arecollectively referred to as “genome sequences of MaACS-1˜12”),respectively. These polynucleotides and proteins may be obtained by themethods described in EXAMPLES below, known genetic engineeringtechniques, known methods for synthesis, and so on.

1. Polynucleotide of the Invention

First, the present invention provides the polynucleotide described inany one selected from the group consisting of (a) to (g) below:

(a) a polynucleotide comprising any one nucleotide sequence selectedfrom the group consisting of the ORF sequences of MaACS-1˜12;

(b) a polynucleotide comprising any one nucleotide sequence selectedfrom the group consisting of the cDNA sequences of MaACS-1˜12;

(c) a polynucleotide encoding a protein consisting of any one amino acidsequence selected from the group consisting of the amino acid sequencesof MaACS-1˜12;

(d) a polynucleotide encoding a protein consisting of an amino acidsequence wherein 1 to 100 amino acids are deleted, substituted, insertedand/or added in any one amino acid sequence selected from the groupconsisting of the amino acid sequences of MaACS-1˜12, and having anacyl-CoA synthetase activity or an activity of increasing the amountand/or changing the composition, of the fatty acids produced in a hostcell when expressed in the host cell;

(e) a polynucleotide encoding a protein having an amino acid sequencehaving at least 60% identity to any one amino acid sequence selectedfrom the group consisting of the amino acid sequences of MaACS-1˜12, andhaving an acyl-CoA synthetase activity or an activity of increasing theamount and/or changing the composition, of the fatty acids produced in ahost cell when expressed in the host cell; and,

(f) a polynucleotide which hybridizes to a polynucleotide consisting ofa nucleotide sequence complementary to any one nucleotide sequenceselected from the group consisting of the ORF sequences of MaACS-1˜12under stringent conditions, and which encodes a protein having anacyl-CoA synthetase activity or an activity of increasing the amountand/or changing the composition, of the fatty acids produced in a hostcell when expressed in the host cell; and,.

(g) a polynucleotide which hybridizes to a polynucleotide consisting ofa nucleotide sequence complementary to any one nucleotide sequenceselected from the group consisting of the cDNA sequences of MaACS-1˜12under stringent conditions, and which encodes a protein having anacyl-CoA synthetase activity or an activity of increasing the amountand/or changing the composition, of the fatty acids produced in a hostcell when expressed in the host cell.

As used herein, the term “polynucleotide” means a DNA or RNA.

As used herein, the term “polynucleotide which hybridizes understringent conditions” refers to a polynucleotide obtained by the colonyhybridization method, plaque hybridization method, Southernhybridization method or the like, using as a probe, for example, apolynucleotide consisting of a nucleotide sequence complementary to anyone nucleotide sequence selected from the group consisting of the ORFsequences of MaACS-1˜12 or any one nucleotide sequence selected from thegroup consisting of the cDNA sequences of MaACS-1˜12, or the whole orpart of a polynucleotide consisting of the nucleotide sequence encodingany one amino acid sequence selected from the group consisting of theamino acid sequences of MaACS-1˜12. For the methods of hybridization,there are used the methods described in, e.g., “Sambrook & Russell,Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor,Laboratory Press 2001,” “Ausubel, Current Protocols in MolecularBiology, John Wiley & Sons 1987-1997,” etc.

As used herein, the term “stringent conditions” may be any of lowstringent conditions, moderate stringent conditions and high stringentconditions. The term “low stringent conditions” are, for example, 5×SSC,5× Denhardt's solution, 0.5% SDS, 50% formamide at 32° C. The term“moderate stringent conditions” are, for example, 5×SSC, 5× Denhardt'ssolution, 0.5% SDS, 50% formamide at 42° C., or 5×SSC, 1% SDS, 50 mMTris-HCl (pH 7.5), 50% formamide at 42° C. The term “high stringentconditions” are, for example, 5×SSC, 5× Denhardt's solution, 0.5% SDS,50% formamide at 50° C. or 0.2×SSC, 0.1% SDS at 65° C. Under theseconditions, a DNA with higher identity is expected to be obtainedefficiently at higher temperatures, though multiple factors are involvedin hybridization stringency including temperature, probe concentration,probe length, ionic strength, time, salt concentration and others, and aperson skilled in the art may appropriately select these factors toachieve similar stringency.

When commercially available kits are used for hybridization, forexample, an Alkphos Direct Labeling and Detection System (GE Healthcare)may be used. In this case, according to the attached protocol, aftercultivation with a labeled probe overnight, the membrane is washed witha primary wash buffer containing 0.1% (w/v) SDS at 55° C. to detect thehybridized DNA. Alternatively, in producing a probe based on thenucleotide sequence complementary to any one nucleotide sequenceselected from the group consisting of the ORF sequences of MaACS-1˜12 orany one nucleotide sequence selected from the group consisting of thecDNA sequences of MaACS-1˜12, or based on the entire or part of thenucleotide sequence encoding any one amino acid sequence selected fromthe group consisting of the amino acid sequences of MaACS-1˜12,hybridization can be detected with a DIG Nucleic Acid Detection Kit(Roche Diagnostics) when the probe is labeled with digoxigenin (DIG)using a commercially available reagent (e.g., a PCR Labeling Mix (RocheDiagnostics), etc.).

In addition to those described above, other polynucleotides that can behybridized include DNAs having 50% or higher, 51% or higher, 52% orhigher, 53% or higher, 54% or higher, 55% or higher, 56% or higher, 57%or higher, 58% or higher, 59% or higher, 60% or higher, 61% or higher,62% or higher, 63% or higher, 64% or higher, 65% or higher, 66% orhigher, 67% or higher, 68% or higher, 69% or higher, 70% or higher, 71%or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher,76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% orhigher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85%or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher,90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% orhigher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99%or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% orhigher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% orhigher or 99.9% or higher identity with the DNA for any one nucleotidesequence selected from the group consisting of the ORF sequences ofMaACS-1˜12 or for any one nucleotide sequence selected from the groupconsisting of the cDNA sequences of MaACS-1˜12, or with the DNA encodingany one amino acid sequence selected from the group consisting of theamino acid sequences of MaACS-1˜12, as calculated by a homology searchsoftware, such as FASTA, BLAST, etc. using default parameters.

Identity between amino acid sequences or nucleotide sequences may bedetermined using FASTA (Science 227 (4693): 1435-1441, (1985)),algorithm BLAST (Basic Local Alignment Search Tool) by Karlin andAltschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990; Proc. Natl.Acad. Sci. USA, 90: 5873, 1993). Programs called blastn, blastx, blastp,tblastn and tblastx based on the BLAST algorithm have been developed(Altschul S. F. et al., J. Mol. Biol. 215: 403, 1990). When a nucleotidesequence is sequenced using blastn, the parameters are, for example,score=100 and wordlength=12. When an amino acid sequence is sequencedusing blastp, the parameters are, for example, score=50 andwordlength=3. When BLAST and Gapped BLAST programs are used, defaultparameters for each of the programs are employed.

The polynucleotides of the present invention described above can beobtained by known genetic engineering techniques or known methods forsynthesis.

2. Protein of the Invention

The present invention provides the proteins shown below.

(i) A protein encoded by the polynucleotide of any one of (a) to (g)above.

(ii) A protein comprising any one amino acid sequence selected from thegroup consisting of the amino acid sequences of MaACS-1˜12.

(iii) A protein consisting of an amino acid sequence wherein one or moreamino acids are deleted, substituted, inserted and/or added in any oneamino acid sequence selected from the group consisting of the amino acidsequences of MaACS-1˜12, and having an acyl-CoA synthetase activity oran activity of increasing the amount and/or changing the composition, ofthe fatty acids produced in a host cell when expressed in the host cell.

(iv) A protein having an amino acid sequence having at least 90%identity to any one amino acid sequence selected from the groupconsisting of the amino acid sequences of MaACS-1˜12, and having anacyl-CoA synthetase activity or an activity of increasing the amountand/or changing the composition, of the fatty acids produced in a hostcell when expressed in the host cell.

The proteins described in (iii) or (iv) above are typically naturallyoccurring mutants of the protein consisting of any one amino acidsequence selected from the group consisting of the amino acid sequencesof MaACS-1˜12 and include those proteins which may be artificiallyobtained using site-directed mutagenesis described in, e.g., “Sambrook &Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold SpringHarbor Laboratory Press 2001,” “Ausubel, Current Protocols in MolecularBiology, John Wiley & Sons 1987-1997,” “Nuc. Acids. Res., 10, 6487(1982),” “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982),” “Gene, 34, 315(1985),” “Nuc. Acids. Res., 13, 4431 (1985),” “Proc. Natl. Acad. Sci.USA, 82, 488 (1985),” etc.

As used herein, the “protein consisting of an amino acid sequencewherein one or several acids are deleted, substituted, inserted and/oradded in any one amino acid sequence selected from the group consistingof the amino acid sequences of MaACS-1˜12, and having an acyl-CoAsynthetase activity or an activity of increasing the amount and/orchanging the composition, of the fatty acids produced in a host cellwhen expressed in the host cell” includes proteins consisting of anamino acid sequence wherein, e.g., 1 to 100, 1 to 90, 1 to 80, 1 to 70,1 to 60, 1 to 50, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35,1 to 34, 1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27,1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19,1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11,1 to 10, 1 to 9 (1 to several), 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4,1 to 3, 1 to 2, or one amino acid is/are deleted, substituted, insertedand/or added in any one amino acid sequence selected from the groupconsisting of the amino acid sequences of MaACS-1˜12, and having theacyl-CoA synthetase activity or the activity of increasing the amountand/or changing the composition, of the fatty acids produced in a hostcell when expressed in the host cell. In general, the number ofdeletions, substitutions, insertions, and/or additions is preferablysmaller.

Such proteins include a protein having an amino acid sequence having theidentity of approximately 60% or higher, 61% or higher, 62% or higher,63% or higher, 64% or higher, 65% or higher, 66% or higher, 67% orhigher, 68% or higher, 69% or higher, 70% or higher, 71% or higher, 72%or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher,77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% orhigher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86%or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher,91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% orhigher, 96% or higher, 97% or higher, 98% or higher, 99% or higher,99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher,99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or99.9% or higher, to any one amino acid sequence selected from the groupconsisting of the amino acid sequences of MaACS-1˜12, and having thediacylglycerol acyltransferase activity. As the identity percentagedescribed above is higher, the protein is preferable in general.

The term deletion, substitution, insertion and/or addition of one ormore amino acid residues in the amino acid sequence of the protein ofthe invention is intended to mean that one or more amino acid residuesare deleted, substituted, inserted and/or added at optional and one ormore positions in the same sequence. Two or more types of deletions,substitutions, insertions and additions may occur at the same time.

Examples of the amino acid residues which are mutually substitutable aregiven below. Amino acid residues in the same group are mutuallysubstitutable. Group A: leucine, isoleucine, norleucine, valine,norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine,t-butylglycine, t-butylalanine and cyclohexylalanine; Group B: asparticacid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipicacid and 2-aminosuberic acid; Group C: asparagine and glutamine; GroupD: lysine, arginine, ornithine, 2,4-diaminobutanoic acid and2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline and4-hydroxyproline; Group F: serine, threonine and homoserine; and GroupG: phenylalanine and tyrosine.

The protein of the present invention may also be produced by chemicalsynthesis methods such as the Fmoc method (fluorenylmethyloxycarbonylmethod), the tBoc method (t-butyloxycarbonyl method), etc. In addition,peptide synthesizers available from Advanced Automation Peptide ProteinTechnologies, Perkin Elmer, Protein Technologies, PerSeptive, AppliedBiosystems, SHIMADZU Corp., etc. may also be used for the chemicalsynthesis.

The protein encoded by the polynucleotide of the invention and theprotein of the invention are both ACS homolog proteins and considered tohave the acyl-CoA synthetase activity since the ATP-AMP motif andFACS/VLACS-FATP motif, which are important for the acyl-CoA synthetaseactivity, are conserved. As used herein, ATP, AMP, FACS, VLACS and FATPare intended to mean adenosine triphosphate, adenosine monophosphate,fatty acyl-CoA synthetase, very long chain acyl-CoA synthetase and fattyacid transport protein, respectively. Specific amino acid sequences ofthe ATP-AMP motif and FACS/VLACS-FATP motif contained in the protein ofthe present invention are shown in FIGS. 25 and 26 at the singleunderlined and double underlined sequences, respectively. With regard torepresentative amino acid sequences of the ATP-AMP motif andFACS/VLACS-FATP motif, reference may be made to databases including pfam(http://pfam.sanger.ac.uk/), etc.

As used herein, the term “acyl-CoA synthetase activity (ACS activity)”is intended to mean the activity of promoting the acyl-CoA-formingreaction through formation of a thioester bond between a fatty acid andcoenzyme A (chemical reaction equation below).Fatty acid+Coenzyme A→Acyl-CoA+H₂O

The acyl-CoA synthetase activity can be quantitatively confirmed, forexample, by cultivating for a certain period of time host cells, intowhich the polypeptide of the present invention is introduced, preparingthe lysate of the host cells, mixing the cell lysate with a labeledfatty acid (e.g., polyunsaturated fatty acid labeled with a radioactiveisotope, etc.) and coenzyme A, reacting them for a certain period oftime, then extracting free fatty acids with n-heptane, and quantifyingthe fatty acyl-CoA which is formed during the above reaction andremained in the aqueous fraction, using a scintillation counter. Fordetails of the method for confirming the acyl-CoA synthetase activity,reference may be made to Black P. N., et al. (J. B. C., 272 (8),4896-4903, 1997). Alternatively, the acyl-CoA synthetase activity mayalso be assayed by the method described in “Evaluation of ACS Activity”of EXAMPLE 2, which involves no radioactive label.

The “activity of increasing the amount of the fatty acids produced in ahost cell when expressed in the host cell” is intended to mean theactivity that, when the polynucleotide of the present invention or thepolynucleotide encoding the protein of the present invention isintroduced (transformed) into a host cell and expressed in the hostcell, increases the total fatty acid production, as compared to areference cell (control) derived from the same strain as the host cellin which the polynucleotide described above is not introduced.

The “activity of changing the composition of the fatty acids produced ina host cell when expressed in the host cell” is intended to mean theactivity that, when the polynucleotide of the present invention or thepolynucleotide encoding the protein of the present invention isintroduced (transformed) into a host cell and expressed in the hostcell, changes the amount or ratio of various fatty acids produced, ascompared to a reference cell (control) derived from the same strain asthe host cell in which the polynucleotide described above is notintroduced.

As used herein, the term “fatty acid” is intended to mean an aliphaticmonocarboxylic acid (a carboxylic acid having one carboxylic residue andcarbon atoms connected to each other in a chain) represented by generalformula RCOOH (wherein R is an alkyl). The fatty acid includes asaturated fatty acid having no double bond and an unsaturated fatty acidcontaining a double bond(s) in the hydrocarbon chain. The fatty acid ispreferably an unsaturated fatty acid, and more preferably, apolyunsaturated fatty acid containing a plurality of double bonds in thehydrocarbon chain. The polyunsaturated fatty acid includes preferably anunsaturated fatty acid having carbon atoms of 18 or more, e.g., anunsaturated fatty acid having carbon atoms of 18 or 20, and examplesinclude, but not limited to, oleic acid, linoleic acid, linolenic acid(γ-linolenic acid, dihomo-γ-linolenic acid, etc.), arachidonic acid, andthe like. The polyunsaturated fatty acids are particularly preferablylinoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid and arachidonicacid, more preferably, linoleic acid, dihomo-γ-linolenic acid andarachidonic acid, and most preferably, dihomo-γ-linolenic acid andarachidonic acid.

In the present invention, the “host cell” is not particularly limited solong as the cell is capable of expressing the polynucleotide of theinvention when the polynucleotide is introduced. The cells include cellsderived from mammals (excluding human), insects, plants, fungi,bacteria, etc., preferably cells from plants and fungi, more preferably,cells from fungi, and most preferably, lipid-producing fungi or yeast.

The lipid-producing fungi which can be used are the lipid-producingfungi described in, e.g., MYCOTAXON, Vol. XLIV, No. 2, pp. 257-265(1992). Specific examples include, but not limited to, microorganismsbelonging to the genus Mortierella including microorganisms belonging tothe subgenus Mortierella, e.g., Mortierella elongata IFO8570,Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, Mortierellaalpina IFO8568, ATCC16266, ATCC32221, ATCC42430, CBS 219.35, CBS224.37,CBS250.53, CBS343.66, CBS527.72, CBS528.72, CBS529.72, CBS608.70 andCBS754.68, etc., or microorganisms belonging to the subgenus Micromucor,e.g., Mortierella isabellina CBS194.28, IFO6336, IFO7824, IFO7873,IFO7874, IFO8286, IFO8308 and IFO7884, Mortierella nana IFO8190,Mortierella ramanniana IFO5426, IFO8186, CBS112.08, CBS212.72, IFO7825,IFO8184, IFO8185 and IFO8287, Mortierella vinacea CBS236.82, etc. Amongothers, Mortierella alpina is preferable.

Specific examples of the yeast include the genus Saccharomyces, thegenus Candida, the genus Zygosaccharomyces, the genus Pichia and thegenus Hansenula, and preferably, Saccharomyces cerevisiae in the genusSaccharomyces. In wild strains of yeast such as Saccharomycescerevisiae, etc., saturated fatty acids or monovalent fatty acids havingmainly 18 or less carbon atoms can be synthesized within the cells, butpolyunsaturated fatty acids cannot be synthesized therein. For thisreason, when yeast such as Saccharomyces cerevisiae, etc. is used as ahost cell, it is preferred to impart the ability to synthesizepolyunsaturated fatty acids to the yeast cells by genetic engineering,etc. The ability to synthesize polyunsaturated fatty acids can beimparted by introducing a gene encoding a protein derived from anorganism that already possesses the ability to synthesizepolyunsaturated fatty acids and takes part in fatty acid synthesis.

The “organism that already possesses the ability to synthesizepolyunsaturated fatty acids” includes, for example, lipid-producingfungi. Specific examples of the lipid-producing fungi are the same asthose given hereinabove.

Examples of the gene encoding a protein derived from an organism thatalready possesses the ability to synthesize polyunsaturated fatty acidsand “gene encoding the protein that takes part in fatty acid synthesis”include, but not limited to, Δ12 fatty acid desaturase gene, Δ6 fattyacid desaturase gene, GLELO fatty acid elongase gene and Δ5 fatty aciddesaturase gene, etc. The nucleotide sequences of Δ12 fatty aciddesaturase gene, Δ6 fatty acid desaturase gene, GLELO fatty acidelongase gene and Δ5 fatty acid desaturase gene are available by havingaccess to databases including GenBank, etc. For example, in GenBank,Accession No. AB020033, No. AB020032, No. AB193123 and No. AB188307 areentered to access the respective sequences.

The genes for fatty acid synthesis-related proteins described above areinserted into appropriate vectors (e.g., pESC (Stratagene), pYES(Invitrogen), etc.), which are then introduced into yeast by theelectroporation method, the spheroplast method (Proc. Natl. Acad. Sci.USA, 75 p1929 (1978)), the lithium acetate method (J. Bacteriology, 153,p163 (1983)), and the methods described in Proc. Natl. Acad. Sci. USA,75 p1929 (1978), Methods in Yeast Genetics, 2000 Edition: A Cold SpringHarbor Laboratory Course Manual, etc.

Fatty acids can be extracted from the host cells transformed by thepolynucleotide of the present invention or the polynucleotide encodingthe protein of the present invention in the following manner. A hostcell is cultured and then treated in a conventional manner, e.g., bycentrifugation, filtration, etc. to obtain cultured cells. The cells arethoroughly washed with water and preferably dried. Drying may beaccomplished by lyophilization, air-drying, etc. Depending uponnecessity, the dried cells are disrupted using a Dynomil or byultrasonication, and then extracted with an organic solvent preferablyin a nitrogen flow. Examples of the organic solvent include ether,hexane, methanol, ethanol, chloroform, dichloromethane, petroleum etherand so on. Alternatively, good results can also be obtained byalternating extraction with methanol and petroleum ether or byextraction with a single-phase solvent system ofchloroform-methanol-water. Removal of the organic solvent from theextract by distillation under reduced pressure may give fattyacid-containing lipids. The fatty acids extracted may be converted intothe methyl esters by the hydrochloric acid methanol method, etc.

The quantity or ratio of various fatty acids may be determined byanalyzing the fatty acids extracted as described above using variouschromatography techniques. Examples of the chromatography techniquesinclude, but not limited to, high performance liquid chromatography andgas chromatography, and particularly preferably, gas chromatography.

3. Vector of the Invention and Vector-Introduced Transformants

In another embodiment, the present invention further provides theexpression vector comprising the polynucleotide of the invention.

The vector of the invention is generally constructed to contain anexpression cassette comprising:

(i) a promoter that can be transcribed in a host cell;

(ii) any of the polynucleotides defined in (a) to (g) above that islinked to the promoter; and,

(iii) an expression cassette comprising as a component a signal thatfunctions in the host cell with respect to the transcription terminationand polyadenylation of RNA molecule.

The vector thus constructed is introduced into a host cell. Examples ofhost cells which may be appropriately used in the present invention arethe same as described above.

In these host cells transformed by the vector of the present invention,the ACS activity is more increased, fatty acids are more produced or thequantity or ratio of various fatty acids contained in the cells arechanged, when compared to the host cells which are not transformed bythe vector of the present invention.

Examples of the vectors available for introducing into lipid-producingfungi include, but not limited to, pDura5 (Appl. Microbiol. Biotechnol.,65, 419-425, (2004)).

Any vector is available as the vector used to introduce into the yeastand not particularly limited so long as it is a vector capable ofexpressing the insert in the yeast cells. The vector includes, e.g.,pYE22m (Biosci. Biotech. Biochem., 59, 1221-1228, 1995).

Promoters/terminators for regulating gene expression in host cells maybe used in an optional combination as far as they function in the hostcells. For example, a promoter of the histone H4.1 gene, a promoter ofthe glyceraldehyde-3-phosphate dehydrogenase, etc. may be used.

As selection markers used for the transformation, there may be utilizedauxotrophic markers (ura5, niaD), hygromycin-resistant gene,zeocin-resistant gene, genecitin-resistant gene (G418r),copper-resistant gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA,81, 337 1984), cerulenin-resistant gene (fas2m, PDR4) (Junji Inokoshi,et al., Biochemistry, 64, 660, 1992; and Hussain et al., Gene, 101: 149,1991, respectively), and the like.

For the transformation of host cells, generally known methods may beused. In lipid-producing fungi, the transformation may be performed,e.g., by the electroporation method (Mackenzie, D. A. et al., Appl.Environ. Microbiol., 66, 4655-4661, 2000) and the particle deliverymethod (the method described in JPA 2005-287403 “Method of BreedingLipid-Producing Fungus”). On the other hand, the electroporation method,the spheroplast method (Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)) andthe lithium acetate method (J. Bacteriology, 153 p163 (1983)) as well asthe methods described in Proc. Natl. Acad. Sci. USA, 75 p1929 (1978),Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor LaboratoryCourse Manual, etc) may be used for the transformation of yeast.However, the method for transformation is not limited to those describedabove.

For general cloning techniques, reference may be made to “Sambrook &Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold SpringHarbor Laboratory Press 2001”, “Methods in Yeast Genetics, A laboratorymanual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),”etc.

4. Method for Producing the Lipid or Fatty Acid Composition of theInvention

In another embodiment, the present invention further provides a methodfor preparing a lipid or fatty acid composition which comprises usingthe transformant described above.

As used herein, the term “lipid” is intended to mean a simple lipidincluding a compound (e.g., a glyceride) which is composed of a fattyacid and an alcohol attached via an ester linkage, or its analog (e.g.,a cholesterol ester), etc.; a complex lipid in which phosphoric acid,amino acid(s), saccharide(s) or the like are bound to a part of thesimple lipid; or a derived lipid which is a hydrolysate of the lipid andis insoluble in water.

As used herein, the term “oil and fat” is intended to mean an ester ofglycerol and a fatty acid (glyceride).

The term “fatty acid” is the same as defined above.

The method for extracting the lipid or fatty acid composition of thepresent invention is the same as the method for extracting fatty acidsdescribed above.

Fatty acids can be separated from the above fatty acid-containing lipidsin a state of mixed fatty acids or mixed fatty acid esters byconcentration and separation in a conventional manner (e.g., ureaaddition, separation under cooling, column chromatography, etc.).

The lipids produced by the method of the present invention includepreferably unsaturated fatty acids, and more preferably, polyunsaturatedfatty acids. Preferred examples of the polyunsaturated fatty acids areunsaturated fatty acids having 18 or more carbon atoms, e.g.,unsaturated fatty acids having 18 to 20 carbon atoms, and include, butnot limited to, oleic acid, linoleic acid, linolenic acid (γ-linolenicacid and dihomo-γ-linolenic acid, etc.), arachidonic acid, etc.Particularly preferred polyunsaturated fatty acids are linoleic acid,γ-linoleic acid, dihomo-γ-linoleic acid and arachidonic acid, morepreferably, linoleic acid, dihomo-γ-linoleic acid and arachidonic acid,and most preferably, dihomo-γ-linolenic acid and arachidonic acid.

The lipids produced by the method of the present invention and thecomposition of the fatty acids contained in the lipids may be confirmedby the lipid extraction method or fatty acid separation method describedabove, or a combination thereof.

The lipid or fatty acid composition obtained by the production method ofthe present invention can be provided for use in producing, e.g., foodproducts, pharmaceuticals, industrial materials (raw materials forcosmetics, soaps, etc.), which contain oils and fats, in a conventionalmanner.

In a still other embodiment, the present invention provides a method forpreparing food products, cosmetics, pharmaceuticals, soaps, etc. usingthe transformant of the present invention. The method involves the stepof forming lipids or fatty acids using the transformant of the presentinvention.

Food products, cosmetics, pharmaceuticals, soaps, etc. containing thelipids or fatty acids produced are prepared in a conventional manner. Assuch, the food products, cosmetics, pharmaceuticals, soaps, etc.produced by the method of the present invention contain the lipids orfatty acids produced using the transformant of the present invention.The present invention further provides the food products, cosmetics,pharmaceuticals, soaps, etc. produced by such a method.

The form of the cosmetic (composition) or pharmaceutical (composition)of the present invention is not particularly limited and may be any formincluding the state of a solution, paste, gel, solid or powder. Thecosmetic composition or pharmaceutical composition of the presentinvention may also be used as cosmetics or topical agents for the skin,including an oil, lotion, cream, emulsion, gel, shampoo, hair rinse,hair conditioner, enamel, foundation, lipstick, face powder, facialpack, ointment, perfume, powder, eau de cologne, tooth paste, soap,aerosol, cleansing foam, etc., an anti-aging skin care agent,anti-inflammatory agent for the skin, bath agent, medicated tonic, skinbeauty essence, sun protectant, or protective and improving agent forskin troubles caused by injury, chapped or cracked skin, etc.

The cosmetic composition of the present invention may further beformulated appropriately with other oils and fats and/or dyes,fragrances, preservatives, surfactants, pigments, antioxidants, etc., ifnecessary. The formulation ratio of these materials may be appropriatelydetermined by those skilled in the art, depending upon purpose (forexample, oils and fats may be contained in the composition in 1 to 99.99wt %, preferably, 5 to 99.99 wt %, and more preferably, 10 to 99.95wt%). If necessary, the pharmaceutical composition of the presentinvention may also contain other pharmaceutically active components(e.g., anti-inflammatory components) or aid components (e.g., lubricantsor vehicle components). Examples of the other components commonly usedin a cosmetic or a skin preparation for external use include an agentfor acne, an agent for preventing dandruff or itching, an antiperspirantand deodorant agent, an agent for burn injury, an anti-mite and liceagent, an agent for softening keratin, an agent for xeroderma, anantiviral agent, a percutaneous absorption promoting agent, and thelike.

The food product of the present invention includes a dietary supplement,health food, functional food, food product for young children, babyfood, infant modified milk, premature infant modified milk, geriatricfood, etc. As used herein, the food or food product is intended to meana solid, fluid and liquid food as well as a mixture thereof, andcollectively means an edible stuff.

The term dietary supplement refers to food products enriched withspecific nutritional ingredients. The term health food refers to foodproducts which are healthful or beneficial to health, and encompassesdietary supplements, natural foods, diet foods, etc. The term functionalfood refers to a food product for replenishing nutritional ingredientswhich assist body control functions and is synonymous with a food forspecified health use. The term food for young children refers to a foodproduct given to children up to about 6 years old. The term geriatricfood refers to a food product treated to facilitate digestion andabsorption when compared to untreated foods. The term infant modifiedmilk refers to modified milk given to children up to about one year old.The term premature infant modified milk refers to modified milk given topremature infants until about 6 months after birth.

The form of these food products includes natural foods (treated withfats and oils) such as meat, fish and nuts; foods supplemented with fatsand oils during cooking, e.g., Chinese foods, Chinese noodles, soups,etc.; foods prepared using fats and oils as heating media, e.g., tempuraor deep-fried fish and vegetables, deep-fried foods, fried bean curd,Chinese fried rice, doughnuts, Japanese fried dough cookies or karinto;fat- and oil-based foods or processed foods supplemented with fats andoils during processing, e.g., butter, margarine, mayonnaise, dressing,chocolate, instant noodles, caramel, biscuits, cookies, cakes, icecream; and foods sprayed or coated with fats and oils upon finishing,e.g., rice crackers, hard biscuits, sweet bean paste bread, etc.However, the food product is not limited to foods containing fats andoils, and other examples include agricultural foods such as bakeryproducts, noodles, cooked rice, sweets (e.g., candies, chewing gums,gummies, sweet tablets, Japanese sweets), bean curd or tofu andprocessed products thereof; fermented foods such as Japanese rice wineor sake, medicinal liquor, sweet cooking sherry or mirin, vinegar, soysauce and bean paste or miso, etc.; livestock food products such asyoghurt, ham, bacon, sausage, etc.; seafood products such as minced andsteamed fish cake or kamaboko, deep-fried fish cake or ageten and puffyfish cake or hanpen, etc.; as well as fruit drinks, soft drinks, sportsdrinks, alcoholic beverages, tea, etc.

The food product of the present invention may also be in the form ofpharmaceutical preparations such as capsules, etc., or in the form of aprocessed food such as natural liquid diets, defined formula diets andelemental diets formulated with the oil and fat of the present inventiontogether with proteins, sugars, trace elements, vitamins, emulsifiers,aroma chemicals, etc., health drinks, enteral nutrients, and the like.

As described above, fatty acids can be efficiently produced byexpressing the ACS homolog gene of the present invention in host cells.

Furthermore, the expression level of the gene can be used as anindicator to study conditions for cultivation, cultivation control, etc.for efficient fatty acid production.

EXAMPLES

Hereinafter, the present invention is described in more detail withreference to EXAMPLES but it should be understood that the invention isnot deemed to limit the scope of the invention to these EXAMPLES.

Example 1

Genome Analysis of M. alpina

The M. alpina 1S-4 strain was plated on 100 ml of GY2:1 medium (2%glucose and 1% yeast extract, pH 6.0) followed by shake culture at 28°C. for 2 days. The mycelial cells were collected by filtration, andgenomic DNA was prepared using DNeasy (QIAGEN). The nucleotide sequenceof the genomic DNA described above was determined using a Roche 454 GSFLX Standard. On this occasion, nucleotide sequencing of a fragmentlibrary was performed in two runs and nucleotide sequencing of a matepaired library in three runs. The resulting nucleotide sequences wereassembled to give 300 supercontigs.

Synthesis of cDNA and Construction of cDNA Library

The M. alpina strain 1S-4 was plated on 100 ml of medium (1.8% glucose,1% yeast extract, pH 6.0) and precultured for 3 days at 28° C. A 10 Lculture vessel (Able Co., Tokyo) was charged with 5 L of medium (1.8%glucose, 1% soybean powder, 0.1% olive oil, 0.01% Adekanol, 0.3% KH₂PO₄,0.1% Na₂SO₄, 0.05% CaCl₂.2H₂O and 0.05% MgCl₂.6H₂O, pH 6.0), and thewhole amount of the pre-cultured product was plated thereon, followed byaerobic spinner culture under conditions of 300 rpm, 1 vvm and 26° C.for 8 days. On Days 1, 2 and 3 of the cultivation, glucose was added inan amount corresponding to 2%, 2% and 1.5%, respectively. The mycelialcells were collected at each stage on Days 1, 2, 3, 6 and 8 of thecultivation to prepare total RNA by the guanidine hydrochloride/CsClmethod. Using an Oligotex-dT30<Super>mRNA Purification Kit (Takara BioInc.)(“dT30” disclosed as SEQ ID NO: 130), poly(A)+RNA was purified fromthe total RNA. A cDNA library was constructed for each stage using aZAP-cDNA Gigapack III Gold Cloning Kit (STRATAGENE).

Search for ACS Homolog

Using as a query the amino acid sequences of ScFAA1 (YOR317W), ScFAA2(YER015W), ScFAA3 (YIL009W), ScFAA4 (YMR246W), ScFAT1 (YBR041W) andScFAT2 (YBR222C), which are ACS from yeast, a tblastn search wasperformed against the genome nucleotide sequence of the M. alpina strain1S-4. As a result, hits were found in twelve (12) sequences. That is,hit was found on supercontigs containing the sequence shown by SEQ IDNO: 5, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQID NO: 30, SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 50,SEQ ID NO: 55 or SEQ ID NO: 60. The genes bearing SEQ ID NO: 5, SEQ IDNO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQID NO: 35, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 50, SEQ ID NO: 55and SEQ ID NO: 60 were designated respectively as MaACS-1, MaACS-2,MaACS-3, MaACS-4, MaACS-5, MaACS-6, MaACS-7, MaACS-8, MaACS-9, MaACS-10,MaACS-11 and MaACS-12.

Cloning of ACS Homolog

For cloning of the cDNAs corresponding to the MaACS-1˜12 genes,screening of the cDNA library described above was performed. Probelabeling was performed by PCR using an ExTaq (Takara Bio Inc.). That is,digoxigenin (DIG)-labeled amplified DNA probes were prepared using a PCRLabeling Mix (Roche Diagnostics) instead of dNTP mix attached to ExTaq.

Conditions for hybridization were set as follows.

-   Buffer: 5×SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formaldehyde;-   Temperature: 42° C. (overnight);-   Wash conditions: 0.2×SSC, in 0.1% SDS solution (65° C.) for 20    mins.×3

Detection was performed using a DIG Nucleic Acid Detection Kit (RocheDiagnositcs). Phage clones were obtained by screening and plasmids wereexcised

from the phage clones by in vivo excision to give the respective plasmidDNAs. Primers for preparing the probes used for screening of therespective genes, the number of nucleotides in CDS of the respectivegenes, the number of amino acids in the amino acid sequences deducedfrom the nucleotide sequences of CDS, and the number of exons andintrons by comparison of genomic DNA sequences with CDS sequences aregiven below.

(1) MaACS-1 (SEQ ID NO: 61) Primer ACS-1-1F:5′-GTCGGCTCCAAGCTTGCAATCC-3′ (SEQ ID NO: 62) Primer ACS-1-2R:5′-GGACAGCTCCAGCACTGTGGTAAAG-3′

cDNA (SEQ ID NO: 4)

CDS (SEQ ID NO: 3): 1857 bp

ORF (SEQ ID NO: 1): 1854 bp

Amino acid sequence (SEQ ID NO: 2): 618 amino acids (see FIG. 1)

Number of exons: 5, number of introns: 4 (see FIG. 2)

(2) MaACS-2 (SEQ ID NO: 63) Primer ACS-2-1F:5′-GACCACGGGATTCCCCAAGGCTGC-3′ (SEQ ID NO: 64) Primer ACS-2-2R:5′-CTTGGTCGCGCTTGTTCCTGGCCAC-3′

cDNA (SEQ ID NO: 9)

CDS (SEQ ID NO: 8): 1929 bp

ORF (SEQ ID NO: 6): 1926 bp

Amino acid sequence (SEQ ID NO: 7): 642 amino acids (see FIG. 3)

Number of exons: 8, number of introns: 7 (see FIG. 4)

(3) MaACS-3 (SEQ ID NO: 65) Primer ACS-3-1F:5′-TACAGCTTTGTTGCTGTCCCCATC-3′ (SEQ ID NO: 66) Primer ACS-3-2R:5′-GATGATGGGTGTGCTTGCAAAGATC-3′

cDNA (SEQ ID NO: 14)

CDS (SEQ ID NO: 13): 1653 bp

ORF (SEQ ID NO: 11): 1650 bp

Amino acid sequence (SEQ ID NO: 12): 550 amino acids (see FIG. 5)

Number of exons: 9, number of introns: 8 (see FIG. 6)

(4) MaACS-4 (SEQ ID NO: 67) Primer ACS-4-1F:5′-AACCCAAAGCTGCGCCAGGCTGTCC-3′ (SEQ ID NO: 68) Primer ACS-4-2R:5′-TTACAGCTTGGATTCCTTTTGATGG-3′

cDNA (SEQ ID NO: 19)

CDS (SEQ ID NO: 18): 2067 bp

ORF (SEQ ID NO: 16): 2064 bp

Amino acid sequence (SEQ ID NO: 17): 688 amino acids (see FIG. 7)

Number of exons: 7, number of introns: 6 (see FIG. 8)

(5) MaACS-5 (SEQ ID NO: 69) Primer ACS-5-1F:5′-GTCGTGCCCGATGCGGAGACGC-3′ (SEQ ID NO: 70) Primer ACS-5-2R:5′-TCAGTGGATCCCGTTATACATCAG-3′

cDNA (SEQ ID NO: 24)

CDS (SEQ ID NO: 23): 1980 bp

ORF (SEQ ID NO: 21): 1977 bp

Amino acid sequence (SEQ ID NO: 22): 659 amino acids (see FIG. 9)

Number of exons: 6, number of introns: 5 (see FIG. 10)

(6) MaACS-6 (SEQ ID NO: 71) Primer ACS-6-1F:5′-GCGTCCCCCTCTATGATACATTG-3′ (SEQ ID NO: 72) Primer ACS-6-2R:5′-GTGGGATGCAGGACGGCAACATCG-3′

cDNA (SEQ ID NO: 29)

CDS (SEQ ID NO: 28): 1980 bp

ORF (SEQ ID NO: 26): 1977 bp

Amino acid sequence (SEQ ID NO: 27): 659 amino acids (see FIG. 11)

Number of introns: at least 5 (see FIG. 12)

(7) MaACS-7 (SEQ ID NO: 73) Primer ACS-7-1F:5′-GGATGCCGAACAACAGCGCGTGG-3′ (SEQ ID NO: 74) Primer ACS-7-2R:5′-GCACCCTCCTCAGAAACAGCCCTC-3′

cDNA (SEQ ID NO: 34)

CDS (SEQ ID NO: 33): 1827 bp

ORF (SEQ ID NO: 31): 1824 bp

Amino acid sequence (SEQ ID NO: 32): 608 amino acids (see FIG. 13)

Number of exons: 5, number of introns: 4 (see FIG. 14)

(8) MaACS-8 (SEQ ID NO: 75) Primer ACS-8-1F:5′-CAGTCGAGTACATTGTCAACCACG-3′ (SEQ ID NO: 76) Primer ACS-8-2R:5′-GCGGTTCAAGAGGCGAGGCACAGC-3′

cDNA (SEQ ID NO: 39)

CDS (SEQ ID NO: 38): 2079 bp

ORF (SEQ ID NO: 36): 2076 bp

Amino acid sequence (SEQ ID NO: 37): 692 amino acids (see FIG. 15)

Number of exons: 8, number of introns: 7 (see FIG. 16)

(9) MaACS-9 (SEQ ID NO: 77) Primer ACS-9-1F:5′-GTTCATCTTCTGCTGGCTGGGTCTC-3′ (SEQ ID NO: 78) Primer ACS-9-2R:5′-GTTGCGTTGTTCACGCGGCAATCC-3′

cDNA (SEQ ID NO: 44)

CDS (SEQ ID NO: 43): 1851 bp

ORF (SEQ ID NO: 41): 1848 bp

Amino acid sequence (SEQ ID NO: 42): 616 amino acids (see FIG. 17)

Number of exons: 5, number of introns: 4 (see FIG. 18)

(10) MaACS-10 (SEQ ID NO: 79) Primer ACS-10-1F:5′-ATGGAAACCTTGGTTAACGGAAAG-3′ (SEQ ID NO: 80) Primer ACS-10-2R:5′-TCAGCAAAGATGGCCTTGGGCTGG-3′

cDNA (SEQ ID NO: 49)

CDS (SEQ ID NO: 48): 2076 bp

ORF (SEQ ID NO: 46): 2073 bp

Amino acid sequence (SEQ ID NO: 47): 691 amino acids (see FIG. 19)

Number of exons: 8, number of introns: 7 (see FIG. 20)

(11) MaACS-11 (SEQ ID NO: 81) Primer ACS-11-1F:5′-GTCAAGGGCGAGACTCGCATCC-3′ (SEQ ID NO: 82) Primer ACS-11-2R:5′-CGGTGACGATGGTCATGGACTGC-3′

cDNA (SEQ ID NO: 54)

CDS (SEQ ID NO: 53): 2043 bp

ORF (SEQ ID NO: 51): 2040 bp

Amino acid sequence (SEQ ID NO: 52): 680 amino acids (see FIG. 21)

Number of exons: 3, number of introns: 2 (see FIG. 22)

(12) MaACS-12 (SEQ ID NO: 83) Primer ACS-12-1F:5′-GCGAGACCCGCATCCGCCGCTCC-3′ (SEQ ID NO: 84) Primer ACS-12-2R:5′-GACCGTCCTCGCCCAGGGTGTCG-3′

cDNA (SEQ ID NO: 59)

CDS (SEQ ID NO: 58): 2043 bp

ORF (SEQ ID NO: 56): 2040 bp

Amino acid sequence (SEQ ID NO: 57): 680 amino acids (see FIG. 23)

Number of exons: 3, number of introns: 2 (see FIG. 24)

Sequencing Analysis

The identity between the CDS nucleotide sequences of 12 ACS homologsfrom M. alpina is shown in TABLE 1 and the identity between the aminoacid sequences is shown in TABLE 2. MaACS-11 and MaACS-12 showed highidentity of 80.2% in the nucleotide sequence and 84.3% in the amino acidsequence.

TABLE 1 Sequence identity among CDS nucleotide sequences of ACS homologsfrom M. alpina MaACS-1 MaACS-2 MaACS-3 MaACS-4 MaACS-5 MaACS-6 MaACS-1 —51.3 42.9 45.4 44.7 46.6 MaACS-2 — 43.4 46.8 46.9 45.7 MaACS-3 — 38.038.2 38.9 MaACS-4 — 50.4 51.6 MaACS-5 — 70.8 MaACS-6 — MaACS-7 MaACS-8MaACS-9 MaACS-10 MaACS-11 MaACS-12 MaACS-7 MaACS-8 MaACS-9 MaACS-10MaACS-11 MaACS-12 MaACS-1 46.0 45.6 69.5 44.7 46.2 45.8 MaACS-2 46.544.6 52.5 44.9 44.9 44.0 MaACS-3 43.7 37.5 42.8 41.5 39.0 39.1 MaACS-443.8 57.7 44.2 47.0 49.7 49.3 MaACS-5 44.7 53.0 44.9 46.6 48.9 47.2MaACS-6 46.2 53.0 45.2 47.9 49.2 49.4 MaACS-7 — 44.2 45.9 42.3 45.0 44.6MaACS-8 — 44.3 48.1 50.7 50.8 MaACS-9 — 42.7 46.2 47.8 MaACS-10 — 51.852.1 MaACS-11 — 80.2 MaACS-12 —

TABLE 2 Sequence identity among amino acid sequences of ACS homologsfrom M. alpina MaACS-1 MaAGS-2 MaACS-3 MaACS-4 MaACS-5 MaACS-6 MaACS-1 —36.6 11.8 13.9 15.1 15.8 MaACS-2 — 11.0 14.0 15.4 15.0 MaACS-3 — 21.721.5 20.8 MaACS-4 — 37.5 37.5 MaACS-5 — 77.9 MaACS-6 — MaACS-7 MaACS-8MaACS-9 MaACS-10 MaACS-11 MaACS-12 MaACS-7 MaACS-8 MaACS-9 MaACS-10MaACS-11 MaACS-12 MaACS-1 18.0 14.8 71.9 13.5 14.6 15.0 MaACS-2 17.213.2 37.0 12.3 12.7 13.8 MaACS-3 13.1 21.1 10.5 17.7 18.5 17.9 MaACS-417.0 50.9 15.4 22.8 29.8 29.5 MaACS-5 17.0 41.2 16.4 25.2 29.1 29.8MaACS-6 16.6 39.8 16.6 25.3 29.9 29.4 MaACS-7 — 15.5 17.0 15.3 16.2 16.7MaACS-8 — 15.2 24.9 27.8 28.6 MaACS-9 — 14.1 14.5 14.7 MaACS-10 — 32.832.6 MaACS-11 — 84.3 MaACS-12 —

Using as query sequences the putative amino acid sequences for the CDSsequences of MaACS-1˜12, BLASTp search was performed against the aminoacid sequences registered in GenBank. The proteins having the amino acidsequence which matched the putative amino acid sequences of MaACS-1˜12with highest score and the identity between these proteins and theputative amino acid sequences of MaACS-1˜12 are shown in TABLE 3. Theidentity of the putative amino acid sequences of MaACS-1˜12 with theamino acid sequences of S. cerevisiae-derived acyl-CoA synthetases arealso shown in TABLE 4.

TABLE 3 Sequence identity between the amino acid sequences of M. alpina-derived ACS homologs and known amino acid sequences identity(%) giMaACS-1 41.8 71014575 Putative protein from Ustilago maydis MaACS-2 35.471014575 Putative protein from Ustilago maydis MaACS-3 23.5 71895089Chick ACS long-chain family member 5 MaACS-4 36.9 115487304 Putativeprotein from Oryza sativa MaACS-5 42.5 168065128 Putative protein fromPhyscomitrella patens MaACS-6 40.9 13516481 Long-chain acyl-CoAsynthetase from Arabidopsis thaliana MaACS-7 45.7 120612991 Putativeprotein from Acidovorax avenae subsp. citrulli MaACS-8 40.0 13516481Long-chain acyl-CoA synthetase from Arabidopsis thaliana MaACS-9 37.867538044 Putative protein from Aspergillus nidulans MaACS-10 33.2171682488 Putative protein from Podospora Anserina MaACS-11 48.8169854433 Putative protein from Coprinopsis atramentarius MaACS-12 45.1156045509 Putative protein from Sclerotinia sclerotiorum

TABLE 4 Comparison of amino acid sequences of M. alpina-derived ACShomologs and amino acid sequences of S. cerevisiae-derived ACS ScFAA1ScFAA2 ScFAA3 ScFAA4 ScFAT1 ScFAT2 MaACS-1 13.8 15.3 13.6 13.5 29.8 18.1MaACS-2 12.5 13.6 13.4 13.5 26.3 17.5 MaACS-3 15.8 14.0 15.0 14.8 13.612.9 MaACS-4 26.3 28.3 23.9 24.2 14.0 16.0 MaACS-5 25.6 28.2 25.5 25.813.2 18.6 MaACS-6 25.3 28.4 25.8 25.5 13.0 18.1 MaACS-7 16.5 17.5 16.016.9 16.6 20.6 MaACS-8 23.0 28.0 21.3 22.8 12.2 14.8 MaACS-9 15.6 15.514.3 14.7 30.1 18.3 MaACS-10 30.8 20.6 30.6 30.6 14.0 14.2 MaACS-11 39.622.6 37.3 38.7 12.9 15.8 MaACS-12 41.3 22.3 39.8 39.0 14.4 16.2

FIG. 25 shows the alignment between MaACS from MaACS-1˜12, which haverelatively high amino acid sequence homology to the S.cerevisiae-derived FAA proteins, and the FAA proteins. FIG. 26 shows thealignment of the ACS homologs having relatively high amino acid sequencehomology to S. cerevisiae-derived FAT proteins. The regions of theATP-AMP motif and FACS/VLACS-FATP motif, which are important motifs forthe ACS activity, are highly conserved in both groups shown in FIGS. 25and 26.

Construction of Expression Vector

Vectors for expressing MaACS-1, MaACS-10, MaACS-11, MaACS-6, MaACS-8 andMaACS-9, respectively, in yeast were constructed as follows, using theexpression vector pYE22m (Biosci. Biotech. Biochem., 59, 1221-1228,1995).

The plasmid containing SEQ ID NO: 29, which was obtained by screeningMaACS-6, was digested with restriction enzymes BamHI and XhoI. Theresulting DNA fragment of approximately 2.1 kbp was ligated to the DNAfragment obtained by digestion of vector pYE22m with restriction enzymesBamHI and SalI using a Ligation High (TOYOBO) to give plasmid pYE-ACS-6.

Using the plasmid containing cDNA of MaACS-8 as a template, PCR wasperformed with the primers below using ExTaq (Takara Bio Inc.). The thusamplified DNA fragment was cloned by a TOPO-TA Cloning Kit (Invitrogen).

Primer EcoRI-ACS-8-F: (SEQ ID NO: 85)5′-GGATCCATGCCTTCCTTCAAAAAGTACAACC-3′ Primer SmaI-ACS-8-R: (SEQ ID NO:86) 5′-CCCGGGCAAAGAGTTTTCTATCTACAGCTT-3′

The nucleotide sequence of the insert was verified and the plasmidcontaining the correct nucleotide sequence was digested with restrictionenzymes EcoRI and SmaI. Using a Ligation High (TOYOBO), the resultingDNA fragment of approximately 2.1 kbp was ligated to the DNA fragmentobtained by digesting vector pYE22m with restriction enzymes EcoRII andSmaI to give plasmid pYE-ACS-8.

Using the plasmid containing cDNA of MaACS-9 as a template, PCR wasperformed with the primers below using ExTaq (Takara Bio Inc.). The thusamplified DNA fragment was cloned by a TOPO-TA Cloning Kit (Invitrogen).

Primer EcoRI-ACS-9-F: (SEQ ID NO: 87) 5′-GAATTCATGGTTGCTCTCCCACTCG-3′Primer BamHI-ACS-9-R: (SEQ ID NO: 88) 5′-GGATCCCTACTATAGCTTGGCCTTGCC-3′

The nucleotide sequence of the insert was verified and the plasmidcontaining the correct nucleotide sequence was digested with restrictionenzymes EcoRI and BamHI. Using a Ligation High (TOYOBO), the resultingDNA fragment of approximately 2.0 kbp was ligated to the DNA fragmentobtained by digesting vector pYE22m with restriction enzymes EcoRII andBamHI to give plasmid pYE-ACS-9.

Using the plasmid containing cDNA of MaACS-1 as a template, PCR wasperformed with the primers below using ExTaq (Takara Bio Inc.). The thusamplified DNA fragment was cloned by a TOPO-TA Cloning Kit (Invitrogen).

Primer EcoRI-ACS-1-F: (SEQ ID NO: 89)5′-GGATCCATGTATGTCGGCTCCAAGCTTGC-3′ Primer SalI-ACS-1-R: (SEQ ID NO: 90)5′-GTCGACTCAAAGCCTGGCTTTGCCGCTGACG-3′

The nucleotide sequence of the insert was verified and the plasmidcontaining the correct nucleotide sequence was digested with restrictionenzymes EcoRI and SalI. Using a Ligation High (TOYOBO), the resultingDNA fragment of approximately 1.9 kbp was ligated to the DNA fragmentobtained by digesting vector pYE22m with restriction enzymes EcoRI andSalI to give plasmid pYE-ACS-1.

Using the plasmid containing cDNA of MaACS-10 as a template, PCR wasperformed with the primers below using ExTaq (Takara Bio Inc.). The thusamplified DNA fragment was cloned by a TOPO-TA Cloning Kit (Invitrogen).

Primer ACS-10-1F: (SEQ ID NO: 91) 5′-GGATCCATGGAAACCTTGGTTAACGGAAAG-3′Primer KpnI-ACS-10-R: (SEQ ID NO: 92)5′-GGTACCTAGAACTTCTTCCACATCTCCTC-3′

The nucleotide sequence of the insert was verified and the plasmidcontaining the correct nucleotide sequence was digested with restrictionenzymes EcoRI and KpnI. Using a Ligation High (TOYOBO), the resultingDNA fragment of approximately 2.1 kbp was ligated to the DNA fragmentobtained by digesting vector pYE22m with restriction enzymes EcoRI andKpnI. Plasmid pYE-ACS-10 was obtained by screening for the orientationthat the GAPDH promoter of vector pYE22m was located at its 5′ end ofCDS of MaACS-10.

Using the plasmid containing cDNA of MaACS-11 as a template, PCR wasperformed with the primers below using ExTaq (Takara Bio Inc.). The thusamplified DNA fragment was cloned by a TOPO-TA Cloning Kit (Invitrogen).

Primer SacI-ACS-11-F: (SEQ ID NO: 93)5′-GAGCTCATGCCAAAGTGCTTTACCGTCAACG-3′ Primer BamHI-ACS-11-R: (SEQ ID NO:94) 5′-GGATCCTTACTTGGAGCCATAGATCTGCTTG-3′

The nucleotide sequence of the insert was verified and the plasmidcontaining the correct nucleotide sequence was digested with restrictionenzymes Sad and BamHI. Using a Ligation High (TOYOBO), the resulting DNAfragment of approximately 2.0 kbp was ligated to the DNA fragmentobtained by digesting vector pYE22m with restriction enzymes Sad andBamHI to give plasmid pYE-ACS-11.

Expression in Yeast

Acquisition of Transformants

The yeast S. cerevisiae EH13-15 strain (trp1, MATα) (Appl. Microbiol.Biotechnol., 30, 515-520, 1989) was transformed with plasmids pYE22m,pYE-MaACS-6, pYE-MaACS-8 and pYE-MaACS-9, respectively, by the lithiumacetate method. The transformants were screened for the ability to growon SC-Trp agar medium (2% agar) (per liter, 6.7 g Yeast Nitrogen Basew/o Amino Acids (DIFCO), 20 g glucose, 1.3 g amino acid powders (amixture of 1.25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3g glutamic acid, 0.6 g histidine, 1.8 g leucine, 0.9 g lysine, 0.6 gmethionine, 1.5 g phenylalanine, 11.25 g serine, 0.9 g tyrosine, 4.5 gvaline, 6 g threonine and 0.6 g uracil).

Cultivation of Yeast

One each from the transformants obtained using the respective plasmidswas provided for the following cultivation experiment.

One platinum loop of the yeast was plated on 10 ml of SC-Trp andcultured with shaking for preincubation at 30° C. for a day. After 1 mlof the preincubation was added to the SC-Trp medium, main cultivationwas performed by shake culturing at 30° C. for a day.

Analysis of Fatty Acids in Mycelia

The yeast culture broth was centrifuged to recover the mycelial cells.After washing with 10 ml of sterile water, the mycelial cells were againcentrifuged, recovered and lyophilized The fatty acids in the mycelialcells were converted into the methyl esters by the hydrochloricacid-methanol method followed by extraction with hexane. After hexanewas removed by distillation, the fatty acids were analyzed by gaschromatography.

The fatty acid production per medium is shown in TABLE 5. In the strainstransformed by ppYE-MaACS-6, pYE-MaACS-8 or pYE-MaACS-9, the fatty acidproduction per medium was increased as compared to the control which wastransformed by pYE22m.

TABLE 5 Fatty Acid Production by Transformant per Medium Control MaACS-6MaACS-8 MaACS-9 Fatty acid production 135 159 196 187 (mg/L)Expression in Arachidonic Acid-Producing Yeast(1) Breeding of Arachidonic Acid-Producing Yeast Strains

To breed arachidonic acid-producing yeast strain (S. cerevisiae), thefollowing plasmids were constructed.

First, using the cDNA prepared from M. alpina strain 1S-4 as a template,PCR was performed with ExTaq using the primer pair of Δ12-f and Δ12-r,Δ6-f and Δ6-r, GLELO-f and GLELO-r, or Δ5-f and Δ5-r to amplify the Δ12fatty acid desaturase gene (GenBank Accession No. AB020033) (hereinafter“Δ12 gene”), the Δ6 fatty acid desaturase gene (GenBank Accession No.AB020032) (hereinafter “Δ6 gene”), the GLELO fatty acid elongase gene(GenBank Accession No. AB193123) (hereinafter “GLELO gene”) and the Δ5fatty acid desaturase gene (GenBank Accession No. AB188307) (hereinafter“Δ5 gene”) in the M. alpina strain 1S-4.

Δ12-f: (SEQ ID NO: 95) 5′-TCTAGAATGGCACCTCCCAACACTATTG-3′ Δ12-r: (SEQ IDNO: 96) 5′-AAGCTTTTACTTCTTGAAAAAGACCACGTC-3′ Δ6-f: (SEQ ID NO: 97)5′-TCTAGAATGGCTGCTGCTCCCAGTGTGAG-3′ Δ6-r: (SEQ ID NO: 98)5′-AAGCTTTTACTGTGCCTTGCCCATCTTGG-3′ GLELO-f: (SEQ ID NO: 99)5′-TCTAGAATGGAGTCGATTGCGCAATTCC-3′ GLELO-r: (SEQ ID NO: 100)5′-GAGCTCTTACTGCAACTTCCTTGCCTTCTC-3′ Δ5-f: (SEQ ID NO: 101)5′-TCTAGAATGGGTGCGGACACAGGAAAAACC-3′ Δ5-r: (SEQ ID NO: 102)5′-AAGCTTTTACTCTTCCTTGGGACGAAGACC-3′

These genes were cloned with the TOPO-TA-Cloning Kit. The clones wereconfirmed by their nucleotide sequences. The clones containing thenucleotide sequences of the Δ12 gene, Δ6 gene, GLELO gene and Δ5 genewere designated as plasmids pCR-MAΔ12DS (containing the nucleotidesequence of the Δ12 gene), pCR-MAΔ6DS (containing the nucleotidesequence of the Δ6 gene), pCR-MAGLELO (containing the nucleotidesequence of the GLELO gene) and pCR-MAΔ5DS (containing the nucleotidesequence of the Δ5 gene), respectively.

On the other hand, the plasmid pURA34 (JPA 2001-120276) was digestedwith restriction enzyme HindIII. The resulting DNA fragment ofapproximately 1.2 kb was inserted into the HindIII site of the vector,which was obtained by digesting pUC18 vector (Takara Bio Inc.) withrestriction enzymes EcoRI and SphI, then blunt ending and self ligatingsaid vector. The clone in which the EcoRI site of the vector was locatedat its 5′ end of URA3 was designated as pUC-URA3. Also, the DNA fragmentof approximately 2.2 kb, which was obtained by digesting YEp 13 withrestriction enzymes SalI and XhoI, was inserted into the SalI site ofvector pUC18. The clone in which the EcoRI site of the vector waslocated at its 5′ end of LUE2 was designated as pUC-LEU2.

Next, the plasmid pCR-MAΔ12DS was digested with restriction enzymeHindIII, followed by blunt ending and further digestion with restrictionenzyme XbaI. The resulting DNA fragment of approximately 1.2 kbp wasligated to the DNA fragment of approximately 6.6 kbp, which was obtainedby digesting vector pESC-URA (STRATAGENE) with restriction enzyme SacI,blunt ending and further digesting with restriction enzyme SpeI. Thus,the plasmid pESC-U-Δ12 was obtained. The plasmid pCR-MAΔ6DS was digestedwith restriction enzyme XbaI, followed by blunt ending and furtherdigestion with restriction enzyme HindIII. The resulting DNA fragment ofapproximately 1.6 kbp was ligated to the DNA fragment of approximately 8kbp, which was obtained by digesting the plasmid pESC-U-Δ12 withrestriction enzyme SalI, blunt ending and further digesting withrestriction enzyme HindIII, thereby to give the plasmid pESC-U-Δ12:Δ6.This plasmid was partially digested with restriction enzyme PvuII. Theresulting fragment of approximately 4.2 kb was inserted into the SmaIsite of pUC-URA3 to give the plasmid pUC-URA-Δ12:Δ6.

Also, the plasmid pCR-MAGLELO was digested with restriction enzymes XbaIand SacI. The resulting DNA fragment of approximately 0.95 kbp wasligated to the DNA fragment of approximately 7.7 kbp, which was obtainedby digesting vector pESC-LEU (STRATAGENE) with restriction enzymes XbaIand SacI. Thus, the plasmid pESC-L-GLELO was obtained. The plasmidpCR-MAΔ5DS was digested with restriction enzyme XbaI, followed by bluntending and further digestion with restriction enzyme HindIII. Theresulting DNA fragment of approximately 1.3 kbp was ligated to the DNAfragment of approximately 8.7 kbp, which was obtained by digesting theplasmid pESC-L-GLELO with restriction enzyme ApaI, blunt ending andfurther digesting with restriction enzyme HindIII, thereby to give theplasmid pESC-L-GLELO:Δ5. This plasmid was digested with restrictionenzyme PvuII and the resulting fragment of approximately 3.2 kbp wasinserted into the SmaI site of pUC-LEU2 to give plasmidpUC-LEU-GLELO:Δ5. The Saccharomyces cerevisiae strain YPH499(STRATAGENE) was co-transformed by the plasmid pUC-URA-Δ12:Δ6 andplasmid pUC-LEU-GLELO:Δ5. The transformants were screened for theability to grow on SC-Leu,Ura agar medium. Among the transformants thusobtained, random one strain was designated as the strain ARA3-1. Bycultivating the strain in a galactose-supplemented medium, the strainbecame capable of expressing from the GAL1/10 promoter the Δ12 fattyacid desaturase gene, the Δ6 fatty acid desaturase gene, the GLELO geneand the 45 fatty acid desaturase gene.

(2) Transformation into Arachidonic Acid-Producing Yeast and Analysis

The ARA3-1 strain was transformed by plasmids pYE22m, pYE-ACS-1,pYE-ACS-10 and pYE-ACS-11, respectively. Transformants were screened forthe ability to grow on SC-Trp,Leu,Ura agar medium (2% agar) (per liter,6.7 g Yeast Nitrogen Base w/o Amino Acids (DIFCO), 20 g glucose and 1.3g amino acid powders (a mixture of 1.25 g adenine sulfate, 0.6 garginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 0.9 glysine, 0.6 g methionine, 1.5 g phenylalanine, 11.25 g serine, 0.9 gtyrosine, 4.5 g valine and 6 g of threonine). Random four strains fromthe respective plasmid-transfected strains were used for the subsequentcultivation.

These strains were cultivated at 30° C. for a day in 10 ml of theSC-Trp,Leu,Ura liquid medium described above. One milliliter of theculture was plated on 10 ml of SG-Trp,Leu,Ura liquid medium (per liter,6.7 g Yeast Nitrogen Base w/o Amino Acids (DIFCO), 20 g galactose and1.3 g amino acid powders (a mixture of 1.25 g adenine sulfate, 0.6 garginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 0.9 glysine, 0.6 g methionine, 1.5 g phenylalanine, 11.25 g serine, 0.9 gtyrosine, 4.5 g valine and 6 g threonine) and then cultivated at 15° C.for 6 days. The mycelial cells were collected, washed with water andthen lyophilized After the fatty acids in the dried mycelial cells wereconverted to the methyl esters by the hydrochloric acid-methanol method,the analysis of fatty acids was performed by gas chromatography. Theratio of each PUFA to the total fatty acids in the control straintransformed by plasmid pYE22m, and in the strains transformed by eachACS homolog from Mortierella is shown in TABLE 6.

TABLE 6 % Ratio of PUFA in ACS homolog expression strains fromMortierella control MaACS-1 MaACS-10 MaACS-11 18:2 7.23 ± 0.11 8.15 ±0.29 14.87 ± 0.28  10.57 ± 0.30  18:3 (n − 6) 0.38 ± 0.01 0.44 ± 0.041.67 ± 0.10 0.92 ± 0.07 DGLA 0.41 ± 0.01 0.42 ± 0.02 0.30 ± 0.17 0.33 ±0.03 ARA 0.42 ± 0.01 0.63 ± 0.04 0.47 ± 0.10 0.75 ± 0.10 Average ±Standard Deviation

As shown in TABLE 6, the ratio of fatty acids could be modified byexpressing the ACS homolog from Mortierella. Particularly in theMaACS-11 expression strain, the ratios of arachidonic acid, linoleicacid and γ-linolenic acid were increased by about 1.8 times, about 1.5times and about 2.4 times, respectively, as compared to the controlstrain. In the MaACS-1 expression strain, the ratio of arachidonic acidwas increased by about 1.5 times, as compared to the control strain.Further in the MaACS-10 expression strain, the ratios of linoleic acidand γ-linolenic acid were increased by about 2 times and about 4 times,respectively, as compared to the control strain.

Example 2

Construction of Expression Vector

Expression Vector for Yeast

The vector pYE-ACS-12 for expressing MaACS-12 in yeast was constructedas follows. Using a plasmid containing the cDNA of MaACS-12 as atemplate, PCR was performed with the following primers usingKOD-Plus-(TOYOBO).

Primer Eco-ACS-G-F: (SEQ ID NO: 103) 5′-GAATTCATGACAAAGTGCCTCACCGTCG-3′Primer Sma-ACS-G-R: (SEQ ID NO: 104) 5′-CCCGGGACTTAGGCCGTTCCATAAAGCTG-3′The amplified DNA fragment was cloned using a Zero Blunt TOPO PCRCloning Kit (Invitrogen). The nucleotide sequence of the insert wasverified and the plasmid containing the correct nucleotide sequence wasdigested with restriction enzymes EcoRI and SmaI. Using a Ligation High(TOYOBO), the resulting DNA fragment of approximately 2 kbp was ligatedto the DNA fragment obtained by digesting vector pYE22m with restrictionenzyme BamHI and then blunt ending with a Blunting Kit (TAKARA Bio) andfurther digesting with EcoRI, to give plasmid pYE-ACS-12.Expression Vector for M. alpina

The vector for expressing MaACS-10 and MaACS-11 in M. alpina wasconstructed as follows.

First, pUC18 was digested with restriction enzymes EcoRI and HindIII andan adapter obtained by annealing oligo DNA MCS-for-pUC18-F2 withMCS-for-pUC18-R2 was inserted therein to construct plasmid pUC18-RF2.

MCS-for-pUC18-F2: (SEQ ID NO: 105)5′-AATTCATAAGAATGCGGCCGCTAAACTATTCTAGACTAGGTCGA CGGCGCGCCA-3′MCS-for-pUC18-R2: (SEQ ID NO: 106)5′-AGCTTGGCGCGCCGTCGACCTAGTCTAGAATAGTTTAGCGGCCG CATTCTTATG-3′

Using the genome DNA of M. alpina as a template, PCR was performed withthe primers Not1-GAPDHt-F and EcoR1-Asc1-GAPDHt-R usingKOD-Plus-(Toyobo). The amplified DNA fragment of about 0.5 kbp wascloned using a Zero Blunt TOPO PCR Cloning Kit (Invitrogen). After thenucleotide sequence of the insert was verified, the DNA fragment ofabout 0.9 kbp obtained by digesting with restriction enzymes NotI andEcoRI was inserted into the NotI and EcoRI site of plasmid pUC18-RF2 toconstruct plasmid pDG-1.

Not1-GAPDHt-F: (SEQ ID NO: 107) 5′-AGCGGCCGCATAGGGGAGATCGAACC-3′EcoR1-Asc1-GAPDHt-R: (SEQ ID NO: 108)5′-AGAATTCGGCGCGCCATGCACGGGTCCTTCTCA-3′

Using the genome of M. alpina as a template, PCR was performed with theprimers URASg-F1 and URASg-R1 using KOD-Plus-(Toyobo). The amplified DNAfragment was cloned using a Zero Blunt TOPO PCR Cloning Kit(Invitrogen). After the nucleotide sequence of the insert was verified,the DNA fragment of about 2 kbp obtained by digestion with SalI wasinserted into the SalI site of plasmid pDG-1. The plasmid that the 5′end of URAS gene inserted was oriented toward the EcoRI side of thevector was designated as the plasmid pDuraG.

URA5g-F1: (SEQ ID NO: 109) 5′-GTCGACCATGACAAGTTTGC-3′ URA5g-R1: (SEQ IDNO: 110) 5′-GTCGACTGGAAGACGAGCACG-3′

Subsequently, PCR was performed with KOD-Plus-(TOYOBO) using the genomeof M. alpina as a template and the primers hisHp+URAS-F and hisHp+MGt-EUsing an In-Fusion (registered trade name) Advantage PCR Cloning Kit(TAKARA Bio), the amplified DNA fragment of about 1.0 kbp was ligated tothe DNA fragment of about 5.3 kbp amplified by PCR withKOD-Plus-(TOYOBO) using pDuraG as a template and the primerspDuraSC-GAPt-F and URASgDNA-F, to give plasmid pDUra-RhG.

hisHp + URA5-F: (SEQ ID NO: 111)5′-GGCAAACTTGTCATGAAGCGAAAGAGAGATTATGAAAACAAGC-3′ hisHp + MGt-F: (SEQ IDNO: 112) 5′-CACTCCCTTTTCTTAATTGTTGAGAGAGTGTTGGGTGAGAGT-3′pDuraSC-GAPt-F: (SEQ ID NO: 113) 5′-TAAGAAAAGGGAGTGAATCGCATAGGG-3′URA5gDNA-F: (SEQ ID NO: 114) 5′-CATGACAAGTTTGCCAAGATGCG-3′

Using the plasmid pDUra-RhG as a template, the DNA fragment of about 6.3kbp was amplified by PCR with KOD-Plus-(TOYOBO) using the primerspDuraSC-GAPt-F and pDurahG-hisp-R.

pDurahG-hisp-R: (SEQ ID NO: 115) 5′-ATTGTTGAGAGAGTGTTGGGTGAGAGTG-3′

Using the plasmid containing cDNA of MaACS-10, the DNA fragment of about2.1 kbp was amplified by PCR with KOD-Plus-(TOYOBO), using the primersbelow.

Primer ACS-10 + hisp-F: (SEQ ID NO: 116)5′-CACTCTCTCAACAATATGGAAACCTTGGTTAACGGAAAGT-3′ Primer ACS-10 + MGt-R:(SEQ ID NO: 117) 5′-CACTCCCTTTTCTTACTAGAACTTCTTCCACATCTCCTCAATA TC-3′The resulting DNA fragment was ligated to the 6.3 kbp DNA fragmentdescribed above using an In-Fusion (registered trade name) Advantage PCRCloning Kit (TAKARA BIO) to give plasmid pDUraRhG-ACS-10.

Using the plasmid containing cDNA of MaACS-11 as a template, the 2.1 kbpDNA fragment was amplified by PCR with KOD-Plus-(TOYOBO) using theprimers below.

Primer ACS-11 + MGt-R: (SEQ ID NO: 118)5′-CACTCCCTTTTCTTATTACTTGGAGCCATAGATCTGCTTGA-3′ Primer ACS-11 + hisp-F:(SEQ ID NO: 119) 5′-CACTCTCTCAACAATATGCCAAAGTGCTTTACCGTCAAC-3′

The resulting DNA fragment was ligated to the 6.3 kbp DNA fragmentdescribed above using an In-Fusion (registered trade name) Advantage PCRCloning Kit (TAKARA BIO) to give the plasmid pDUraRhG-ACS-11.

Evaluation of ACS Activity

The yeast EH13-15 was transformed by plasmids pYE22m, pYE-ACS-5,pYE-ACS-8, pYE-ACS-10, pYE-ACS-11 and pYE-ACS-12, respectively, andrandom two transformants obtained were cultivated as follows. Oneplatinum loop of the mycelial cells were plated on 10 ml of SC-Trpmedium and cultivated with shaking for preincubation at 30° C. for aday. After 1% of the preincubation was added to 100 ml of the SD-Trpmedium, main cultivation was performed by shake culturing at 28° C. fora day.

The crude enzyme solution was prepared as follows. The mycelial cellswere collected by centrifugation, washed with water and temporarilystored at −80° C. The mycelial cells were suspended in 5 ml of Buffer B(50 mM sodium sulfate buffer (pH 6.0), 10% glycerol and 0.5 mM PMSF).The mycelial cells were then disrupted with a French press (16 kPa, 3times). Centrifugation was carried out at 1,500×g at 4° C. for 10minutes and centrifuged. The supernatant obtained was used as the crudeenzyme solution.

The ACS activity was determined by the following procedures based on thedescription of a reference literature (J.B.C., 272 (8), 1896-4903,1997). The reaction solution contained 200 mM Tris-HCl (pH7.5), 2.5 mMATP, 8 mM MgCl₂, 2 mM EDTA, 20 mM NaF, 0.1% TritonX-100, 50 μg/ml fattyacids, 50 μM CoA and 100 μl of the crude enzyme solution (suitablydiluted in Buffer B), and was made 500 μl in total. The reaction wascarried out at 28° C. for 30 minutes. After completion of the reaction,2.5 ml of stop solution (isopropanol:n-heptane:1 M sulfuric acid(40:20:1)) was added and the mixture was thoroughly agitated.Furthermore, 2 ml of n-heptane was added thereto. After thoroughlymixing them, the mixture was centrifuged to recover the upper layer.Further 2 ml of n-heptane was added to the lower layer and treated inthe same manner to recover the upper layer. The upper layers recoveredwere combined and evaporated to dryness using a centrifugalconcentrator. Then, 50 μl of 0.2 mg/ml tricosanoic acid (23:0) was addedthereto as an internal standard. The fatty acids were converted into themethyl esters by the hydrochloric acid-methanol method, followed byfatty acid analysis using gas chromatography. The amount of the fattyacids, which were changed to acyl-CoA and thus distributed into thelower layer by the procedures above, was calculated from the amount offatty acids detected. The results are shown in the table below. The ACSactivity is expressed as the amount of fatty acids distributed into thelower layer by the procedures above, per weight of the protein in thecrude enzyme solution. The control is the strain transformed by pYE22mand the others are the transformants in which the expression vectors ofthe respective genes were introduced.

TABLE 7 ACS Activity on Palmitic Acid MaACS- MaACS- MaACS- MaACS- 5 1011 12 Control #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 mg/mg · protein 0.26 0.200.41 0.34 0.49 0.43 0.31 0.40 0.11 0.12

When palmitic acid was used as substrate, MaACS-5, MaACS-10, MaACS-11and MaACS-12 showed the ACS activity of approximately 2 to 4 times thecontrol.

TABLE 8 ACS Activity on Oleic Acid MaACS- MaACS- MaACS- 10 11 12 Control#1 #2 #1 #2 #1 #2 #1 #2 mg/mg · protein 0.25 0.20 0.25 0.16 0.16 0.180.09 0.11

When oleic acid was used as substrate, MaACS-10, MaACS-11 and MaACS-12showed the ACS activity of approximately twice the control.

TABLE 9 ACS Activity on Linoleic Acid MaACS- MaACS- MaACS- MaACS- MaACS-5 8 10 11 12 Control #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 mg/mg · protein0.47 0.42 0.42 0.38 5.24 5.67 3.46 3.20 0.95 0.78 0.14 0.14

When linoleic acid was used as substrate, MaACS-5, MaACS-8 and MaACS-12showed the ACS activity of several times (approximately 3, 3 and 6times, respectively) the control, whereas MaACS-10 and MaACS-11 showedthe ACS activity of several tens times (approximately 40 and 20 times,respectively) the control.

TABLE 10 ACS Activity on γ-Linoleic Acid MaACS- MaACS- MaACS- MaACS-MaACS- 5 8 10 11 12 Control #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 mg/mg ·protein 0.28 0.28 0.16 0.31 0.63 0.59 0.90 0.75 0.52 0.63 0.07 0.09

When γ-linoleic acid was used as substrate, all of MaACS-5, MaACS-8,MaACS-10, MaACS-11 and MaACS-12 showed the ACS activity of approximately2 to 10 times the control.

TABLE 11 ACS Activity on Dihomo-γ-Linoleic Acid MaACS- MaACS- MaACS- 1011 12 Control #1 #2 #1 #2 #1 #2 #1 #2 mg/mg · protein 4.98 4.21 2.752.98 2.04 1.86 0.09 0.05

When dihomo-γ-linoleic acid was used as substrate, all of MaACS-10,MaACS-11 and MaACS-12 showed the ACS activity of several tens times(approximately 60 times, 40 times and 30 times, respectively) thecontrol.

TABLE 12 ACS Activity on Arachidonic Acid MaACS- MaACS- MaACS- 10 11 12Control #1 #2 #1 #2 #1 #2 #1 #2 mg/mg · protein 8.12 7.19 2.73 2.87 1.080.87 0.13 0.03

When arachidonic acid was used as substrate, MaACS-10, MaACS-11 andMaACS-12 showed the ACS activity of several tens times (approximately 90times, 30 times and10 times, respectively) the control.

As above, MaACS-10, MaACS-11 and MaACS-12 in particular showed a higheractivity on polyunsaturated fatty acids of 20 carbon atoms such asdihomo-γ-linoleic acid or arachidonic acid.

Arachidonic Acid Uptake Activity of ACS-Expressed Yeast

The yeast EH13-15 was transformed by plasmids pYE22m, pYE -ACS-10,pYE-ACS-11 and pYE-ACS-12, respectively, and random two transformantsobtained were cultivated as follows. One platinum loop of the cells wereplated on 10 ml of SC-Trp medium and cultivated with shaking forpreincubation at 30° C. for a day. After 100 μl of the preincubation wasadded to 10 ml of the SC-Trp medium in which 50 μg/ml of arachidonicacid was supplemented, main cultivation was performed by shake culturingat 25° C. for a day. The mycelial cells were collected, lyophilized andsubjected to fatty acid analysis. The ratio of arachidonic acid taken upinto the mycelial cells to the added arachidonic acid was determined Theresults are shown in TABLE 14. The control is the strain transformed bypYE22m and the others are the transformants in which the expressionvectors of the respective genes were introduced.

TABLE 13 Dry Mycelial Weight MaACS- MaACS- MaACS- Control 10 11 12 #1 #2#1 #2 #1 #2 #1 #2 % 36.63 37.81 65.86 66.64 61.53 61.35 63.64 67.06

TABLE 14 Ratio of Arachidonic Acid Taken Up into Mycelia MaACS- MaACS-MaACS- Control 10 11 12 #1 #2 #1 #2 #1 #2 #1 #2 mg/10 ml 15.30 15.8019.60 18.10 16.70 17.40 16.80 16.20Acquisition of M. Alpina Transformants

Using as a host the uracil-auxotrophic strain Aura-3 derived from M.alpina strain 1S-4 as described in PCT International PublicationPamphlet WO 2005/019437 entitled “Method of Breeding Lipid-ProducingFungus”), transformation was performed by the particle delivery methodusing the plasmids pDUraRhG-ACS-10 and pDUraRhG-ACS-11, respectively.For screening of the transformants, SC agar medium was used (0.5% YeastNitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco), 0.17%ammonium sulfate, 2% glucose, 0.002% adenine, 0.003% tyrosine, 0.0001%methionine, 0.0002% arginine, 0.0002% histidine, 0.0004% lysine, 0.0004%tryptophan, 0.0005% threonine, 0.0006% isoleucine, 0.0006% leucine,0.0006% phenylalanine, and 2% agar).

Evaluation of M. Alpina Transformants

The transformants obtained were plated on 4 ml of GY medium and culturedwith shaking at 28° C. for 2 days. The mycelial cells were collected byfiltration, and RNA was extracted with an RNeasy Plant Kit (QIAGEN). ASuperScript First Strand System for RT-PCR (Invitrogen) was used tosynthesize cDNA. To confirm expression of the respective genes from theintroduced constructs, RT-PCR was performed with the following primerpairs.

ACS10-RT1: (SEQ ID NO: 120) 5′-GTCCCGAATGGTTCCT-3′ ACS10-RT2: (SEQ IDNO: 121) 5′-AGCGGTTTTCTACTTGC-3′ ACS11-RT1: (SEQ ID NO: 122)5′-AACTACAACCGCGTCG-3′ ACS11-RT2: (SEQ ID NO: 123)5′-CGGCATAAACGCAGAT-3′

In the transformants that overexpression was confirmed, one transformanteach was plated on 10 ml of GY medium (2% glucose and 1% yeast extract)and cultured with shaking at 28° C. at 300 rpm for 3 days. The wholevolume of the culture was transferred to 500 ml of GY medium (2Sakaguchi flask) and shake cultured at 28° C. and 120 rpm. Three, seven,ten and twelve days after this day, 5 ml each and 10 ml each were takenand filtered. After the mycelial cells were dried at 120° C., fattyacids were converted into the methyl esters by the hydrochloricacid-methanol method and analyzed by gas chromatography. The fatty acidproduction and the amount of arachidonic acid produced, per driedmycelial cells were monitored with the passage of time. The transformanthost strain Δura-3 was used as control. The results are shown in FIG.27A and FIG. 27B (MaACS-10) and FIG. 28A and FIG. 28B (MaACS-11).

As shown in FIGS. 27A, 27B, 28A, and 28B, when MaACS-10 and MaACS-11were overexpressed in M. alpina, both the amount of fatty acids and theamount of arachidonic acid per mycelia were increased as compared to thecontrol.

INDUSTRIAL APPLICABILITY

The polynucleotide of the present invention is expressed in anappropriate host cell to efficiently produce fatty acids, in particular,polyunsaturated fatty acids. The fatty acids produced in host cellsaccording to the present invention can be used to produce fatty acidcompositions, food products, cosmetics, pharmaceuticals, soaps, etc.

What is claimed is:
 1. A polynucleotide, which is a cDNA, according toany one selected from t group consisting of (a) to (d) below: (a) apolynucleotide comprising the nucleotide sequence shown by SEQ ID NO:58; (b) a polynucleotide encoding a protein consisting of the amino acidsequence shown by SEQ ID NO: 57; (c) a polynucleotide encoding a proteinconsisting of an amino acid sequence wherein 1 to 10 amino acids aredeleted, substituted, inserted and/or added in the amino acid sequenceshown by SEQ ID NO: 57, and having an acyl-CoA synthetase activity or anactivity of increasing the amount or changing the composition, of thefatty acids produced in a host cell when expressed in the host cell; and(d) a polynucleotide encoding a protein having an amino acid sequencehaving at least 95% identity to the amino acid sequence shown by SEQ IDNO: 57, and having an acyl-CoA synthetase activity or an activity ofincreasing the amount or changing the composition, of the fatty acidsproduced in a host cell when expressed in the host cell.
 2. Thepolynucleotide according to claim 1, comprising the nucleotide sequenceshown by SEQ ID NO:
 58. 3. The polynucleotide according to claim 1,encoding a protein consisting of the amino acid sequence shown by SEQ IDNO:
 57. 4. The polynucleotide according to claim 1, which is a DNA.
 5. Avector comprising the polynucleotide according to claim
 1. 6. Anon-human transformant, into which the polynucleotide according to claim1 is introduced.
 7. The polynucleotide according to claim 2, which is aDNA.
 8. The polynucleotide according to claim 3, which is a DNA.
 9. Avector comprising the polynucleotide according to claim
 2. 10. A vectorcomprising the polynucleotide according to claim
 3. 11. A non-humantransformant, into which the polynucleotide according to claim 2 isintroduced.
 12. A non-human transformant, into which the polynucleotideaccording to claim 3 is introduced.
 13. A non-human transformant, intowhich the vector according to claim 5 is introduced.
 14. A method forproducing a lipid or fatty acid composition, which comprises culturingthe transformant according to claim 6, and collecting the lipid or fattyacid composition from the culture.
 15. The method according to claim 14,wherein the lipid is a triacylglycerol.
 16. The method according toclaim 14, wherein the fatty acid is a polyunsaturated fatty acid havingat least 18 carbon atoms.